This proposal describes a series of studies of the origin, composition, and function of the human in vitro hematopoietic microenvironment. Using adherent stromal cells from long-term marrow cultures as candidate microenvironmental cells, aspects of their growth, origin, hematopoietic function, antigenic characteristics, and abnormalities in disease will be examined. These studies are in part possible due to the development of a new technique for immortalizing these cells using simian virus 40. With this method, clonable microenvironmental cell lines can be produced from most patients and the origin, growth and function can be studied. A unique feature of these cell lines is that they appear to differentiate into cells with hematopoietic as well as stromal characteristics despite rigorous subcloning. This proposal will examine aspects of the control of cell differentiation of these transformed lines. Monoclonal antibodies will be produced that recognize cell surface molecules on stromal cells and then used to evaluate the hematopoietic function of these molecules. Specifically, the ability of these antibodies to impair hematopoietic function of stromal cells will be tested. Growth control of stromal cells and hematopoietic stimulating factor production will be assessed from cell lines adapted to serum-free growth. Differences between transformed and non-transformed stromal cells will be examined on molecular as well as on a functional level. The effect of stromal cells and factors they may produce will be assessed on colony-forming cell progenitors that appear only to proliferate in long-term marrow cultures. Overall, these studies should enhance knowledge of the origin, composition, and function of the hematopoietic microenvironment.